Hsp90 is widely appreciated for its obligatory role in facilitating protein folding. Of the known Hsp90- dependent clients, 48 are directly related to oncogenesis, with Hsp90 clients being represented in all six hallmarks of cancer. Thus, inhibitors of Hsp90 provide a combinatorial attack that simultaneously derail multiple signaling cascades. Consequently, Hsp90 has emerged as an exciting new target for the development of anti-tumor agents. We have recently discovered that curcumin, a natural product found in Indian curry, inhibits Hsp90. In contrast to geldanamycin-derivatives that are currently in clinical trials, curcumin and its analogues can be easily synthesized for elucidation of structure-activity relationships and enhanced affinity for Hsp90. In addition, several other natural products that affect signaling pathways containing Hsp90-dependent proteins have been proposed to manifest their inhibitory activities through Hsp90 inhibition. Therefore, our specific aims are: 1) to identify new natural product inhibitors of Hsp90, libraries of natural products and their derivatives will be screened for their ability to inhibit the Hsp90- dependent refolding of luciferase and Hsp90's ATPase activity using assays developed in our laboratories;2) to elucidate the biochemical mechanism of action of identified inhibitors by the use of in vitro assays that clearly differentiate whether molecules bind to Hsp90's N- or C-terminal ATP binding pocket (assays for Hsp90-dependent client maturation, pull down assays to determine effects on Hsp90/co-chaperone/client interactions, and protease fingerprints);3) to determine the pharmacological properties of inhibitors in cancer cell lines by evaluation in anti-proliferative/ cytotoxicity assays, induction of Hsp90-dependent client protein degradation, and increased Hsp90 levels as hallmarks of Hsp90 inhibition;and 4) to elucidate structure-activity relationships for these inhibitors. Results from this work will identify new natural product inhibitors of Hsp90 and will provide more efficacious drugs that can be easily modified for increased potency against a wide range of cancers, and may lead to the development of more potent and less toxic chemotherapeutic agents. This proposal is innovative because for the first time, we will clearly show that the mechanism of action for natural products (e.g., curcumin) that are currently in clinical trials for the treatment of cancer act through Hsp90 inhibition.